Gene transfer methods

ABSTRACT

Improved methods for transferring a gene into target cells by using a retrovirus, wherein the gene transfer efficiency is improved and the target cells are efficiently transformed by binding the retrovirus to a functional substance which is immobilized on as carrier and having an activity of binding to retroviruses followed by washing; using an antibody capable of specifically recognizing cells, laminin or mannose-rich type sugar chain as a substance having an activity of binding to the target cells; pre-treating the target cells so as to inactivate transferring receptor, or introducing a new functional group into the functional substance.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.10/657,076, filed Sep. 9, 2003, which is a divisional of applicationSer. No. 09/743,354, filed Jan. 9, 2001, which is a 371 national stageof the international application PCT/JP99/03403, filed Jun. 25, 1999.The entire disclosures of the three above-identified applications areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method that increases the efficiencyof gene transfer into target cells and enables efficient transduction ofthe target cells, as well as a series of related techniques therewith,in the fields of medicine, cell technology, genetic engineering,developmental technology and the like.

BACKGROUND ART

Mechanisms of a number of human diseases have been elucidated.Recombinant DNA techniques and the techniques for transferring a geneinto cells have progressed rapidly. Under these circumstances, protocolsfor somatic gene therapies for treating severe genetic diseases havebeen recently developed. More recently, attempts have been made to applygene therapy not only to treatment of genetic diseases but also totreatment of viral infections such as AIDS and cancers.

In most of the gene therapies which have been examined for clinicalapplication the humans to date, a gene is transferred into cells byusing a recombinant retrovirus vector. The retrovirus vector efficientlytransfers the foreign gene of interest into cells and stably integratesthe gene into their chromosomal DNA. Therefore, it is a preferable meansof gene transfer, particularly for gene therapy where long-term geneexpression is desired. Such a vector has been subjected to variousmodifications so as not to have a harmful influence on the organism withthe transferred gene. For example, the replication function of thevector is eliminated such that the vector used for the gene transferdoes not replicate in the cells while repeating unlimited infection(gene transfer).

Since such a vector (a replication-deficient retrovirus vector) cannotautonomously replicate, a retrovirus vector encapsidated in a virusparticle is generally prepared by using retrovirus-producer cells(packaging cells). The simplest method for efficiently transferring agene into target cells comprises co-culturing the target cells with theretrovirus-producer cells. However, retrovirus-producer cells maycontaminate the gene transferred target cells which are to betransplanted to a living body in this method.

Recently, it was reported that the presence of fibronectin, a componentof the extracellular matrix, or a fragment thereof increases theefficiency of gene transfer into cells using a retrovirus (J. Clin.Invest., 93:1451-1457 (1994); Blood, 88:855-862 (1996)). Also, it hasbeen demonstrated that a fibronectin fragment produced by geneticengineering technique has similar properties and can be utilized toefficiently transfer a foreign gene into hematopoietic stem cells (WO95/26200). It is suggested that the binding of a heparin-binding regionin fibronectin to a retrovirus is involved in the increase in genetransfer efficiency due to fibronectin.

Furthermore, it is disclosed in WO 97/18318 that functional substancesother than fibronectin, such as fibroblast growth factor, increase genetransfer efficiency. The publication also discloses that similarincrease in gene transfer efficiency is also observed when a mixture ofa functional substance having an activity of binding to a retrovirus andanother functional substance having an activity of binding to cells isused.

The gene transfer methods using functional substances enable anefficient gene transfer without co-cultivating retrovirus-producer cellsand target cells. It is believed that the increase in gene transferefficiency by the methods is due to the increase in the chance ofinteraction between the retrovirus and the target cells which areclosely co-localized with the aid of the functional substances.

In gene transfer using a retrovirus, target cells are infected with theretrovirus, resulting in gene transfer as described above. However, thegene transfer efficiency using a retrovirus is still unsatisfactory forpractical clinical application. Thus, it is desired to further increaseinfection efficiency.

Increased infection efficiency or gene transfer efficiency may beaccomplished by increasing the concentration (titer) of the retrovirusin the virus suspension (supernatant) used. However, construction andestablishment of virus-producer cells that can produce high titerviruses usually requires much labor. A pseudo-type virus vectorutilizing an envelope protein from vesicular stomatitis virus [Proc.Natl. Acad. Sci. USA, 90:8033-8037 (1993)] can be concentrated bycentrifugation. However, since such concentration of the vector can beused only for this particular vector, it can not be widely used.

Additionally, specific infection of target cells with a retrovirus ingene transfer may achieve high gene transfer efficiency even if thepurity of target cells is low. However, no convenient and efficientmethod is known in the current state of the art.

OBJECTS OF INVENTION

In view of the circumstances described above, the main object of thepresent invention is to provide an improved method for transferring agene into target cells using a retrovirus, in which the gene transferefficiency is increased and the target cells are efficiently transduced.

Hereinafter, other objects and advantages of the present invention willbe explained in detail with reference to the attached drawings.

SUMMARY OF INVENTION

The present inventors have found that gene transfer efficiency isincreased by contacting recombinant retrovirus with a functionalsubstance having an activity of binding to a retrovirus, and beingimmobilized on a substrate with a retrovirus and then washing thesubstrate prior to infecting the target cells.

The present inventors have also found that a gene can be transferredspecifically for target cells of interest and/or efficiently byinfecting the target cells with a recombinant retrovirus in the presenceof a functional substance, such as an antibody which specifically bindsto the target cells, laminin, a sugar chain derived from laminin or ahigh mannose type sugar chain.

The present inventors have further found that gene transfer efficiencycan be increased by appropriately pre-treating target cells beforesubjecting them to gene transfer.

Furthermore, the present inventors have found that the effect on genetransfer of a functional substance having an activity of binding to avirus can be improved by chemically modifying the functional substanceto increase its basicity.

The present invention is completed based on these new findings by thepresent inventors.

Thus, the first aspect of the present invention is a method fortransferring a gene into target cells using a retrovirus, characterizedin that the method comprises:

(1) contacting a solution containing a recombinant retrovirus with afunctional substance having an activity of binding to the retrovirus andbeing immobilized on a substrate;

(2) washing the substrate to which the recombinant retrovirus is bound;and

(3) contacting and incubating the substrate to which the recombinantretrovirus is bound with target cells.

Without limitation, step (1) above is carried out, for example, for 1hour or longer, preferably for 3 hours or longer. In addition, thefrequency of contact between the recombinant retrovirus and thefunctional substance having an activity of binding to the retrovirus maybe physically increased.

Examples of the functional substances having an activity of binding tothe retrovirus which can be used in the present invention include, butare not limited to, fibronectin, fibroblast growth factor, collagen typeV, polylysine and DEAE-dextran, as well as fragments thereof andsubstances having an equivalent activity of binding to the retrovirusthereto. The functional substance may have an activity of binding totarget cells. Alternatively, the functional substance may be used incombination with another functional substance having an activity ofbinding to the target cells. Examples of the functional substanceshaving an activity of binding to the target cells which can be usedinclude, but are not limited to, cell-adhesive proteins, hormones,cytokines, antibodies, sugar chains, carbohydrates and metabolites.

For example, a culture supernatant of retrovirus-producer cells can beused as a source of retrovirus for gene transfer in the presentinvention. The culture supernatant may be obtained in the presence of asubstance that enhances retrovirus production such as sodium butyrate.

The second aspect of the present invention is a method for transferringa gene into target cells using a recombinant retrovirus, characterizedin that the method comprises infecting target cells with a recombinantretrovirus in the presence of two functional substances:

(1) a functional substance having an activity of binding to theretrovirus; and

(2) an antibody which specifically binds to the target cells.

Examples of antibodies which specifically bind to the target cells usedin the present invention include, but are not limited to, an antibodythat recognizes a biological substance on the surface of the targetcells.

The third aspect of the present invention is a method for transferring agene into target cells using a recombinant retrovirus, characterized inthat the method comprises infecting target cells with a recombinantretrovirus in the presence of two functional substances:

(1) a functional substance having an activity of binding to theretrovirus; and

(2) laminin, a laminin fragment, a sugar chain derived from laminin or ahigh mannose type sugar chain.

Examples of the functional substances having an activity of binding tothe retrovirus which can be used in the second and third aspects of thepresent invention include, but are not limited to, fibronectin,fibroblast growth factor, collagen type V, polylysine and DEAE-dextran,as well as fragments thereof and substances having an equivalentactivity of binding to the retrovirus. The functional substance may havean activity of binding to target cells. Furthermore, the functionalsubstance may be used as being immobilized on an appropriate substrate.

The fourth aspect of the present invention is a method for transferringa gene into target cells using a recombinant retrovirus, characterizedin that the method comprises culturing target cells in a medium thatcontains Fe at a low concentration before the target cells are contactedwith the recombinant retrovirus. Examples of culture media which can beused in the present invention include, but are not limited to, a mediumthat contains deferoxamine. Preferably, the method is carried out in thepresence of a functional substance.

The fifth aspect of the present invention relates to a method forincreasing an activity of a peptide or a protein for binding to aretrovirus, characterized in that the method comprises chemicallymodifying the peptide or the protein. Examples of chemical modificationsinclude, but are not limited to, activation of an amino acid residue inthe peptide or the protein and introduction of a basic residue. Forexample, the activation of an amino acid residue is preferably carriedout by treating the peptide or the protein with a water-solublecarbodiimide or with a water-soluble carbodiimide and a diaminocompound, without limitation. The chemically modified peptide or proteinobtained by the method preferably can be used for gene transfer intotarget cells using a retrovirus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a structure of a high mannose type sugar chaincontaining nine mannose residues in the molecule.

FIG. 2 is a graph that shows the gene transfer efficiency (%) achievedby using chemically modified CH-296 in Example 3.

FIG. 3 is a graph that shows the relationship between relative genetransfer efficiency (%) and contact/binding time in a study on theeffect of removing viral infection-inhibitory substances in Example 13.

FIG. 4 is a graph which shows the relationship between relative genetransfer efficiency (%) and respective virus-binding procedures in astudy on the effect of binding retroviruses to functional substancesutilizing the centrifugation method in Example 15.

FIG. 5 is a graph that shows the gene transfer efficiency (%) achievedby using the centrifugation method or the centrifugation-infectionmethod in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

A recombinant retrovirus vector is usually used in the gene transfermethod of the present invention. In particular, a replication-deficientrecombinant retrovirus vector is preferably used. The ability of such avector to replicate is eliminated so that it cannot autonomouslyreplicate in infected cells and, therefore, the vector isnon-pathogenic. The vector can invade a host cell, such as a vertebratecell (particularly, a mammalian cell), and stably integrate a foreigngene inserted within the vector into the chromosomal DNA.

In the present invention, the foreign gene to be transferred into thecells can be inserted into the recombinant retrovirus vector under thecontrol of an appropriate promoter, for example, the LTR promoter in theretrovirus vector or a foreign promoter. In addition, another regulatoryelement (e.g., an enhancer sequence or a terminator sequence) whichcooperates with the promoter and a transcription initiation site mayalso be present in the vector in order to achieve efficienttranscription of the foreign gene. The foreign gene to be transferredmay be a naturally occurring gene or an artificially prepared gene.Alternatively, the foreign gene may be one in which DNA molecules ofdifferent origin are joined together by ligation or other means known inthe art.

One can select any gene, where its transfer into cells is desired, asthe foreign gene to be inserted into the retrovirus vector. For example,a gene encoding an enzyme or a protein associated with the disease to betreated, an intracellular antibody (see, for example, WO 94/02610), agrowth factor, an antisense nucleic acid, a ribozyme, a false primer(see, for example, WO 90/13641) or the like can be used as the foreigngene.

The retrovirus vector used in the present invention may contain asuitable marker gene that enables the selection of gene transferredcells. For example, a drug-resistance gene that confers resistance ofcells to antibiotics or a reporter gene that makes it possible todistinguish the gene transferred cells by detecting its enzymaticactivity can be utilized as the marker gene.

The vectors that can be used in the present invention include, forexample, retrovirus vectors such as MFG vector (ATCC No. 68754), α-SGCvector (ATCC No. 68755) and LXSN vector [BioTechniques, 7:980-990(1989)]. Retrovirus vectors used in the Examples hereinbelow, includingPM5neo vector [Exp. Hematol., 23:630-638 (1995)], contain a neomycinphosphotransferase gene as a marker gene. Thus, cells into which a geneis transferred using the vector can be confirmed based on theirresistance to G418.

These vectors can be prepared as virus particles into which the vectorsare packaged by using a known packaging cell line such as PG13 (ATCCCRL-10686), PG13/LNc8 (ATCC CRL-10685), PA317 (ATCC CRL-9078), GP+E-86(ATCC CRL-9642), GP+envAm12 (ATCC CRL-9641) and φCRIP [Proc. Natl. Acad.Sci. USA, 85:6460-6464 (1988)].

Known media, such as Dulbecco's Modified Eagle's Medium and IscovesModified Dulbecco's Medium, can be used for culturing virus-producercells which are produced by transferring a retrovirus vector intopackaging cells, or for culturing target cells. Such media arecommercially available, for example, from Gibco. Various constituentscan be added to these media depending on the type of target cells usedfor gene transfer or for other objects of the invention. For example,serum or various cytokines can be added to the media in order to promoteor suppress the growth or the differentiation of the target cells. Forexample, calf serum (CS) or fetal calf serum (FCS) can be used as theserum. The cytokine includes interleukins (IL-3, IL-6 etc.),colony-stimulating factors (G-CSF, GM-CSF etc.), stem cell factor (SCF),erythropoietin and various cell growth factors. Many of these cytokinesderived from humans are commercially available. One having the suitableactivity for the objects of the invention is selected from thecytokines. Optionally, the cytokines may be used as a combination ofcytokines.

A sample containing a recombinant retrovirus such as a culturesupernatant of virus-producer cells is used for the gene transfer methodof the present invention. The method for preparing the supernatant isnot limited to a specific method. For example, it is known that additionof sodium butyrate during cultivation of virus-producer cells increasesthe amount of virus particles produced in the supernatant [Human GeneTherapy, 6:1195-1202 (1995)]. The thus prepared high-titer virussupernatant can be used in the gene transfer method of the presentinvention without any problems.

The method of the present invention is characterized in that targetcells are infected with a recombinant retrovirus in the presence of afunctional substance having a retrovirus-binding site. Gene transferredcells can be efficiently obtained by infecting cells with a recombinantretrovirus in the presence of an effective amount of such a functionalsubstance. Furthermore, viral infection-inhibitory substances in a virussupernatant can be readily removed by using the functional substance.Additionally, the presence of a functional substance having an activityof binding to target cells enables gene transfer with higher specificityand/or efficiency.

As used herein, an effective amount is an amount effective to result intransduction of target cells by gene transfer using a recombinantretrovirus. A suitable amount is selected depending on the functionalsubstance to be used and the type of target cells. The amount can bedetermined, for example, by measuring the efficiency of gene transfer bythe method as described herein. As used herein, activities of binding totarget cells include not only an activity of substantially binding tocells but also an activity of keeping in contact with target cells in asolution. The activities can be measured based on the contribution togene transfer efficiency as described above. In addition, gene transferefficiency means the efficiency of transduction.

The above-mentioned functional substances can be used either dissolvedin a solution or immobilized on an appropriate substrate. The substratefor immobilizing a functional substance is not limited to any specificsubstance. Usually, a vessel for cell culture or a bead-shaped substrateis used.

When a functional substance having an activity of binding to a virus andbeing immobilized on a substrate is used, the efficiency of genetransfer can be further increased by using the steps exemplified below.

First, a liquid sample (e.g., a virus supernatant) containing arecombinant retrovirus is contacted with a substrate on which afunctional substance having an activity of binding to a retrovirus isimmobilized. The substrate is washed. The substrate is then directlycontacted with target cells. Alternatively, virus particles eluted fromthe substrate by an appropriate means are added to target cells. Thus, agene can be efficiently transferred. The functional substance having anactivity of binding to a recombinant retrovirus may also have anactivity of binding to target cells. Alternatively, a functionalsubstance having an activity of binding to a recombinant retrovirus anda functional substance having an activity of binding to target cells maybe used in combination.

A step of contacting a liquid sample containing a recombinant retroviruswith a substrate on which a functional substance having an activity ofbinding to the recombinant retrovirus is immobilized is conducted, forexample, for 1 hour or longer, preferably for three hours or longer,without limitation. Also, other conditions including temperature are notspecifically limited. For example, the step can be conducted at roomtemperature or 37° C. Low temperatures around 4° C. may be useddepending on the stability of the virus or the like. The substrate forimmobilizing a functional substance may be appropriately selecteddepending on the object of the invention. If a vessel for cell cultureis used, one can start gene transfer only by adding target cells. Forexample, phosphate buffered saline or Hanks' saline, as the liquidmedium used for culturing target cells or the like, can be used forwashing the substrate.

A retrovirus can be more efficiently bound to a functional substancehaving an activity of binding to the retrovirus by physically increasingthe frequency of contact between the retrovirus and the functionalsubstance. Examples of such physical means include, but are not limitedto, shaking, filtration and centrifugal force. The use of centrifugalforce is specifically exemplified by a method in which a liquid samplecontaining a retrovirus is added to a centrifugation tube in which afunctional substance having an activity of binding to the retrovirus isimmobilized at the bottom and the centrifugation tube is thencentrifuged. The retrovirus is precipitated onto the bottom of thecentrifugation tube by centrifugal force during centrifugation.Accordingly, the frequency of contact between the retrovirus and thefunctional substance having an activity of binding to the retrovirus isincreased, resulting in an increase in the frequency of binding. Theabove-mentioned method does not put the cells under a physical stresslike the method in which viruses are precipitated onto cells bycentrifugal force for purpose of infection (WO 95/10619). Thus, themethod of the present invention results in higher gene transferefficiency.

Gene transfer can be conducted after removing a substance contained in aretrovirus sample, whose presence is not beneficial for gene transfer,by the procedure described above. For example, substances removed by themethod of the present invention include a retroviralinfection-inhibitory substance derived from packaging cells contained ina virus supernatant [Human Gene Therapy, 8:1459-1467 (1997); J. Virol.,70:6468-6473 (1996)], substances added during culturingretrovirus-producer cells in order to enhance retrovirus production suchas phorbol 12-myristate 13-acetate (TPA) and dexamethasone [GeneTherapy, 2:547-551 (1995)], as well as sodium butyrate as describedabove.

Examples of functional substances having an activity of binding to aretrovirus which can be used in the present invention include, but arenot limited to, heparin-II domain of fibronectin, fibroblast growthfactor, collagen type V, polylysine and DEAE-dextran, as well assubstances functionally equivalent to these functional substances (e.g.,a functional substance having a heparin-binding site). Furthermore, amixture of the functional substances, a polypeptide containing thefunctional substance, a polymer of the functional substance, aderivative of the functional substance and the like can be used.

The functional substance's activity of binding to a virus can beenhanced by chemically modifying it. Examples of chemical modificationsinclude activation of an amino acid residue in the functional substanceused and introduction of a basic residue into the substance. Forexample, the activity of binding to a retrovirus can be increased bymodifying a free carboxyl group in a functional substance consisting ofa peptide or a protein with a water-soluble carbodiimide such as1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride to activate thecarboxyl group. Furthermore, the activity of binding to a retrovirus canbe increase by use the thus activated carboxyl group to introduce abasic residue, such as an amino group into the functional substance.

Examples of the functional substance having an activity of binding totarget cells used in the present invention include, but are not limitedto, a substance that has a ligand that binds to the target cells. Theligands include cell-adhesive proteins, hormones or cytokines,antibodies against cell surface antigens, polysaccharides,glycoproteins, glycolipids, sugar chains derived from glycoproteins orglycolipids, and metabolites of the target cells. Furthermore, apolypeptide containing the functional substance, a polymer of thefunctional substance, a derivative of the functional substance, afunctional equivalent of the functional substance or the like can beused.

An antibody that specifically binds to target cells is particularlyuseful for specifically and efficiently transferring a gene intospecific cells. The antibody which can be used in the present inventionis not limited to any specific antibody. An antibody against an antigenexpressed on target cells into which a gene is to be transferred can beappropriately selected for use. Such an antibody can be producedaccording to known methods. Alternatively, many current commerciallyavailable antibodies can also be used. The antibody may be a polyclonalantibody or a monoclonal antibody as long as it has desired propertiessuch as cell specificity. Additionally, an antibody or a derivative ofan antibody modified using known techniques such as a humanizedantibody, a Fab fragment or a single-chain antibody can also be used.

Expression of leukocyte antigens (also known as CD antigens) on variouscells has been studied in detail. Thus, a gene can be transferred intotarget cells with high specificity by selecting an antibody thatrecognizes a CD antigen expressed on the target cells of interest andusing it in the gene transfer method of the present invention. Forexample, gene transfer can be directed to helper T cells by using ananti-CD4 antibody, or to hematopoietic stem cells by using an anti-CD34antibody.

Furthermore, a glycoprotein, laminin, can be used as a functionalsubstance having an activity of binding to target cells to efficientlytransfer a gene into various target cells such as hematopoietic cells.The laminin which can be used in the present invention may be derivedfrom mouse or human, or it may be a fragment thereof as long as it hasan activity of binding to target cells. As described in the examplesbelow, the sugar chain of laminin plays an important role in genetransfer using laminin. Therefore, a sugar chain released from lamininaccording to a known method can also be used in the method of thepresent invention. Furthermore, a glycoprotein having a high mannosetype N-linked sugar chain like laminin, or a sugar chain releasedtherefrom or chemically synthesized, can also be used in the presentinvention. Additionally, a substance such as a protein or the likehaving the above-mentioned sugar chain being attached thereto can beused. For example, a functional substance having an activity of bindingto a retrovirus and having the sugar chain attached thereto canpreferably be used for gene transfer.

The above-mentioned high mannose type sugar chain is not limited to aspecific chain as long as it has 1 to 20 mannose residues in themolecule. A sugar chain having a mannose residue at its non-reducing endis preferably used in the method of the present invention. The sugarchain can be used by being attached to another appropriate molecule suchas a biological molecule (e.g., a monosaccharide, an oligosaccharide, apolysaccharide, an amino acid, a peptide, a protein or a lipid) or anartificial substance such as a synthetic macromolecule.

Representative high mannose type sugar chains derived from organisms areexemplified by those having a structure represented by(Mannose)_(n)-(GlucNAc)₂ [Protein, Nucleic Acid and Enzyme, 43:2631-2639(1998)]. For example, (Mannose)₉-(GlucNAc)₂, a sugar chain which has thestructure as described above and contains nine mannose residues in themolecule, can preferably be used in the gene transfer method of thepresent invention, without limitation (the structure of this sugar chainis shown in FIG. 1).

The functional substance as described above can be obtained fromnaturally occurring substances, an artificial preparation (for example,by recombinant DNA techniques or chemical synthesis techniques), or apreparation that combines a naturally occurring substance and anartificially prepared substance. In addition, a mixture of a functionalsubstance that has a retrovirus-binding site and another functionalsubstance that has a target cell-binding site can be used for the genetransfer using the functional substances as described in WO 97/18318.Alternatively, a functional substance that has a retrovirus-binding siteand a target cell-binding site in a single molecule can be used.Functional substances substantially free of other proteins naturallyassociated the functional substances are used. Additionally, thefunctional substance or a combination of the functional substances canbe combined with a medium used for culturing target cells, cell growthfactor and the like to produce a kit for gene transfer.

Fibronectin or a fragment thereof used in the method of the presentinvention can be prepared in a substantially pure form from naturallyoccurring materials according to methods described, for example, in J.Biol. Chem., 256:7277 (1981); J. Cell. Biol., 102:449 (1986); or J.Cell. Biol., 105:489 (1987). The fibronectin or the fragment thereof canbe prepared using recombinant DNA techniques as described in U.S. Pat.No. 5,198,423. Specifically, a fibronectin fragment containing theheparin-II domain, which is a retrovirus-binding site, such as therecombinant polypeptides including CH-296, H-271, H-296 and CH-271 usedin the Examples below as well as the method for obtaining them aredescribed in detail in the above-mentioned patent. These fragments canbe obtained by culturing Escherichia coli strains deposited underaccession numbers FERM P-10721 (H-296) (the date of the originaldeposit: May 12, 1989), FERM BP-2799 (CH-271) (the date of the originaldeposit: May 12, 1989), FERM BP-2800 (CH-296) (the date of the originaldeposit: May 12, 1989) and FERM BP-2264 (H-271) (the date of theoriginal deposit: Jan. 30, 1989) at the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology,Ministry of International Trade and Industry, 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken as described in the patent publication. Inaddition, fragments that can be typically derived from these fibronectinfragments can be prepared by modifying the plasmids harbored in theEscherichia coli strains above using known recombinant DNA techniques.Among the fibronectin fragments described above, H-296 the bindingregion to VLA-4, CH-271 has the binding region to VLA-5, and CH-296 hasboth [Nature Medicine, 2:876-882 (1996)].

Gene transferred cells can be efficiently obtained by infecting targetcells with a recombinant retrovirus in the presence of the functionalsubstance. The infection with the recombinant retrovirus can be carriedout according to a conventional method, for example, by incubating at37° C. in 5% CO₂. These conditions and the incubation time may besuitably changed depending on the target cells or the objects of theinvention.

Target cells are not infected with a retrovirus when they are in the G₀phase. Therefore, it is preferable to lead the cells into the cell cycleby pre-stimulating them. For this purpose, the target cells are culturedin the presence of a growth factor suitable for the target cells priorto the infection of the cells with the retrovirus. For example, variouscytokines such as interleukin-3, interleukin-6 and stem cell factor areused to pre-stimulate bone marrow cells or hematopoietic stem cells forgene transfer.

It is known that receptors on the surface of cells are involved ininfection of cells with retroviruses. Basic amino acid transporter andphosphate transporter are known to function as receptors for ecotropicviruses and amphotropic viruses, respectively [Proc. Natl. Acad. Sci.USA, 93:11407-11413 (1996)□. It is possible to make target cellssusceptible to viral infection by pre-treating the cells in a medium inwhich concentrations of basic amino acids or phosphates, or salts orprecursors thereof, are reduced in order to activate the expression ormetabolic turnover of the transporters.

Surprisingly, the present inventors have found that activation of thetransferring receptor, whose involvement in viral infection was notpreviously known, also increases the efficiency of retroviral infectionor gene transfer efficiency. The transferring receptor can be activated,without limitation, by treating target cells in a medium containing alimited concentration of Fe. For example, a medium in which Fe ischelated by adding deferoxamine can be used.

Preferably, gene transfer using transferring activation is also carriedout in the presence of the functional substance as described above.

Examples of cells which can be used as the target for gene transfer bythe method of the present invention include, but are not limited to,stem cells, hematopoietic cells, non-adhesive low-density mononuclearcells, adhesive cells, bone marrow cells, hematopoietic stem cells,peripheral blood stem cells, umbilical cord blood cells, fetalhematopoietic stem cells, embryogenic stem cells, embryonic cells,primordial germ cells, oocytes, oogonia, ova, spermatocytes, sperms,CD34+ cells, c-kit+ cells, pluripotent hematopoietic progenitor cells,unipotent hematopoietic progenitor cells, erythroid precursor cells,lymphoid mother cells, mature blood cells, lymphocytes, B cells, Tcells, fibroblasts, neuroblasts, neurocytes, endothelial cells, vascularendothelial cells, hepatocytes, myoblasts, skeletal muscle cells, smoothmuscle cells, cancer cells, myeloma cells, leukemia cells, and so on.The method of the present invention is preferably used for hematopoieticcells which are available from blood and bone marrow because these cellsare relatively easy to obtain and because the techniques for culturingand maintaining them are established. In particular, if long-termexpression of the transferred gene is intended, then blood progenitorcells such as hematopoietic stem cells, CD34-positive cells,c-kit-positive cells and pluripotent hematopoietic progenitor cells aresuitable as target cells.

For example, gene therapy using hematopoietic stem cells as target cellscan be carried out by the following procedure.

First, a material containing hematopoietic stem cells such as bonemarrow tissue, peripheral blood and umbilical cord blood is collectedfrom a donor. Such a material can be directly used in the gene transferprocedure. However, mononuclear cell fractions containing hematopoieticstem cells are usually prepared by means of density-gradientcentrifugation and the like, or hematopoietic stem cells are furtherpurified by utilizing cell surface marker molecules such as CD34 and/orc-kit. The material containing the hematopoietic stem cells is infectedwith a recombinant retrovirus vector, into which a gene of interest isinserted according to the method of the present invention, after beingpre-stimulated by using a suitable cell growth factor, if necessary. Thegene transferred cells thus obtained can be transplanted into arecipient, for example, by intravenous administration. Although therecipient is preferably the donor itself, allogenic transplantation canalso be carried out. For example, if the umbilical cord blood is used asthe material, allogenic transplantation is performed.

Some gene therapies using hematopoietic stem cells as target cells arefor complementing a deficient or abnormal gene in a patient (e.g., genetherapy for ADA deficiency or Gaucher's disease). In addition, a drugresistance gene may be transferred into hematopoietic stem cells inorder to alleviate the damage due to the chemotherapeutic agents usedfor, e.g., the treatment of cancer or leukemia.

Tumor vaccination therapy is investigated as a gene therapy for cancer.In such a therapy, a gene for a cytokine is transferred into cancercells, the cancer cells are deprived of the ability to proliferate, andthe cells are then returned to the body of the patient to enhance tumorimmunity [Human Gene Therapy, 5:153-164 (1994)]. In addition, attemptsare made to treat AIDS using gene therapy. In this case, the followingprocedure is considered. In the procedure, a gene encoding a nucleicacid molecule (e.g., an antisense nucleic acid or a ribozyme) whichinterferes with the replication or the gene expression of HIV (humanimmunodeficiency virus) is transferred into T cells infected with HIV,the causal agent of AIDS [e.g., J. Virol., 69:4045-4052 (1995)].

As described above in detail, a gene can be transferred with highefficiency and with high specificity for target cells by using thepresent invention. Furthermore, the method of the present invention doesnot require any specialized equipment or instrument and is effective forvarious retrovirus vectors and target cells.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Example 1 Preparation of Polypeptides Derived from Fibronectin

A polypeptide derived from human fibronectin, H-271, was prepared fromEscherichia coli HB101/pHD101 (FERM BP-2264) carrying a recombinantplasmid pHD101 which contains a DNA encoding the polypeptide accordingto the method as described in U.S. Pat. No. 5,198,423.

A polypeptide derived from human fibronectin, H-296, was prepared fromEscherichia coli HB101/pHD102 (FERM P-10721) carrying a recombinantplasmid pHD102 which contains a DNA encoding the polypeptide accordingto the method as described in the above-mentioned publication.

A polypeptide CH-271 was prepared as follows.

Briefly, Escherichia coli HB101/pCH101 (FERM BP-2799) was culturedaccording to the method as described in the above-mentioned publication.CH-271 was obtained from the culture.

A polypeptide CH-296 was prepared as follows.

Briefly, Escherichia coli HB101/pCH102 (FERM BP-2800) was culturedaccording to the method as described in the above-mentioned publication.CH-296 was obtained from the culture.

A polypeptide C-274 was prepared as follows.

Briefly, Escherichia coli JM109/pTF7221 (FERM BP-1915) was culturedaccording to the method as described in U.S. Pat. No. 5,102,988. C-274was obtained from the culture.

A polypeptide having an activity of binding to a retrovirus derived fromcollagen type V, ColV, was prepared according to the method as describedin WO 97/18318.

Example 2 Construction of Retrovirus Vector and Preparation ofRetrovirus Supernatant

A retrovirus plasmid, PM5neo vector, which contains a neomycinphosphotransferase gene [Exp. Hematol., 23:630-638 (1995)] wasintroduced into GP+E-86 cells (ATCC CRL-9642). The cells were culturedin Dulbecco's Modified Eagle's Medium (DMEM; Bio Whittaker) containing10% fetal calf serum (FCS; Gibco), 50 units/ml of penicillin and 50μg/ml of streptomycin (both from Gibco). All of the DMEMs used in theprocedure hereinbelow contain the above-mentioned antibiotics. Asupernatant containing PM5neo virus was prepared by adding 4 ml of DMEMcontaining 10% FCS to a plate (a 10-cm gelatin-coated dish for cellculture, Iwaki Glass) in which the above-mentioned producer cells hadbeen grown to semi-confluence, culturing overnight and then collectingthe supernatant. The thus collected culture supernatant was filteredthrough a 0.45-micron filter (Millipore) to prepare a virus supernatantstock, which was stored at −80° C. until use.

Virus supernatants were prepared from the following cells according tothe procedure described above. Ecotropic packaging BOSC23 cells [Proc.Natl. Acad. Sci. USA, 90:8392-8396 (1993)], into which a retrovirusplasmid pLEIN (Clontech; which contains a neomycin phosphotransferasegene and an enhanced green fluorescent protein (EGFP) gene) had beenintroduced; and amphotropic packaging φCRIP cells [Proc. Natl. Acad.Sci. USA, 85:6460-6464 (1988)]. Hereinafter, a virus prepared fromBOSC23 cells is referred to as Eco-EGFP, and a virus prepared from φCRIPcells is referred to as Ampho-EGFP, respectively.

Furthermore, a virus supernatant was prepared from GP+EnvAm12 cells(ATCC CRL-9641) carrying a retrovirus plasmid, TKNeo vector [J. Exp.Med., 178:529-536 (1993)] (which contains a neomycin phosphotransferasegene) according to the procedure as described above. DMEM containing 10%calf serum (CS; Gibco) in place of FCS was used.

The titer of the virus supernatant was measured according to a standardmethod [J. Virol., 62:1120-1124 (1988)] in which the efficiency oftransferring a neomycin phosphotransferase gene into NIH/3T3 cells (ATCCCRL-1658) is used as an index. The number of infectious particlescontained in 1 ml of the supernatant (cfu/ml) was calculated. The amountof the virus supernatant to be added in the experiments hereinbelow wasdetermined based on the calculated value, i.e., the titer of thesupernatant.

Example 3 Preparation of the Functional Substance Having Activity ofBinding to Retrovirus and Measurement of Activity thereof

50 μl of 80 μg/ml solution of H-271, H-296, C-274, CH-271, CH-296, ColV,human basic fibroblast growth factor (bFGF; Progen), tenascin (Gibco) orepidermal growth factor (EGF; Takara Shuzo), or 50 μl of 2% bovine serumalbumin (BSA, Sigma) was added to each well of a 96-well non-treatedmicroplate for cell culture (Falcon). The plate was allowed to stand at4° C. overnight and then washed twice with phosphate buffered saline(PBS; Roman Kogyo). Alternatively, after the plate was treated asdescribed above, 0.1 ml of 4 mg/ml solution of1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (Sigma) insterile pure water was dispensed to each well. The reaction was allowedto proceed at 37° C. for 2 hours. The plate was washed extensively withpure water to prepare a carbodiimide-treated plate. These plates werestored at 4° C. until the viral infection experiments were conducted.

10⁴ mouse leukemia L1210 cells (ATCC CCL-219), which had been grown inRPMI 1640 medium (Bio Whittaker) supplemented with 10% FCS, 50 units/mlpenicillin and 50 μg/ml streptomycin, and 50 μl of PM5neo virussupernatant (10⁴ cfu/ml) were added to the well of the microplate. Afterthe plate was incubated for 24 hours, the medium was changed to the samemedium containing G418 (Gibco) at a final concentration of 0.75 mg/ml,and the plate was then incubated for additional 48 hours.G418-resistance cells were assessed according to the method described inS. Kim et al. [Gene Therapy, 3:1018-1020 (1996)] with a partialmodification in which the color developed using Premix WST-1 reagent(Takara Shuzo) was measured as absorbance at 450 nm. After incubation,10 μl/100 μl culture of the WST-1 reagent was added to the well, theplate was incubated at 37° C. for additional 4 hours. Absorbance at 450nm and 650 nm was then measured using a microplate reader, and thedifference (450 nm−650 nm) was calculated. The value obtained using aplate coated with 2% BSA without carbodiimide treatment was defined asbackground. The results from three rounds of studies are summarized inTable 1. TABLE 1 Functional Treated with substance Untreatedcarbodiimide Experiment 1 BSA 0.000 ± 0.011 Not done CH-271 2.099 ±0.010 2.814 ± 0.079 Experiment 2 BSA 0.000 ± 0.007 0.224 ± 0.031 H-2710.777 ± 0.016 0.994 ± 0.029 H-296 0.474 ± 0.014 0.666 ± 0.021 C-274−0.068 ± 0.017   0.100 ± 0.033 CH-271 0.382 ± 0.017 0.425 ± 0.019 CH-2960.363 ± 0.023 0.460 ± 0.007 ColV 0.644 ± 0.006 0.847 ± 0.033 bFGF 0.425± 0.014 0.580 ± 0.046 Tenascin 0.060 ± 0.021 0.323 ± 0.037 EGF 0.030 ±0.021 0.077 ± 0.038(Mean ± standard deviation)

As shown in Table 1, an increase in gene transfer efficiency wasobserved for known functional substances having an activity of bindingto a virus, i.e., H-271, H-296, CH-271, CH-296, ColV and bFGF.Furthermore, the appearance of G418-resistant cells increased whenC-274, tenascin, EGF and BSA, which do not have an activity of bindingto a virus, were used and the carbodiimide treatment was carried out.

Next, CH-296 was used as a functional substance to carry out experimentsas follows.

0.5 ml of 40 μg/ml CH-296 was added to each well of a 24-wellnon-treated microplate for cell culture (Falcon). The plate wasincubated at 4° C. overnight and then washed with PBS (pH 5.8). 625 μlof 10 mg/ml solution of 1-ethyl-3-dimethylaminopropylcarbodiimidehydrochloride (Sigma) in PBS (pH 5.8) containing ethylenediamine[NH₂(CH₂)₂NH₂; Nacalai Tesque], trimethylenediamine [NH₂(CH₂)₃NH₂;Nacalai Tesque] or putrescine [NH₂(CH₂)₄NH₂; Nacalai Tesque] at avarying concentration was added to each well. The plate was incubated at37° C. for 2 hours. An amino group was introduced to the carboxyl groupin the CH-296 molecule through the mediation of carbodiimide in thisprocedure. The plate was washed three times with PBS, and then blockedwith 2% glycine/PBS followed by 2% BSA/PBS.

GP+E86 cells, into which a retrovirus vector plasmid pLEIN had beenintroduced, were cultured in DMEM containing 10% CS. A supernatant wasthen collected from the culture. 0.5 ml of a virus supernatant preparedby diluting the supernatant to make the concentration to 1×10⁵ cfu/mlwas added to each well of the plate. The plate was incubated for 4hours. 1×10⁴ NIH/3T3 cells were further added to the well. The plate wasincubated for 2 days. After incubation, the cells were collected byusing a cell detachment buffer (Bio Whittaker) and washed.EGFP-expressing cells were analyzed by flow cytometry using FACSVantage™(Becton Dickinson) at an excitation wavelength of 488 nm and an emissionwavelength of 515-545 nm. The binding ability of the virus to the platewas expressed by the efficiency of gene transfer into cells. The resultsare shown in FIG. 2.

As shown FIG. 2, the binding ability of the virus increased as theconcentration of the diamino compound used for introducing an aminogroup increased. About a two-fold increase in the binding ability of thevirus was observed when putrescine, trimethylenediamine orethylenediamine was used in the reaction at a concentration of 2 mM ascompared with the binding ability observed using untreated CH-296.

Example 4 Effect of Enhancing Gene Transfer Efficiency of Laminin

Mouse laminin (Gibco) or human laminin (Takara Shuzo) was used incombination with a functional substance having an activity of binding toa virus to carry out gene transfer experiment. A 24-well non-treatedmicroplate for cell culture (Falcon) used in the experiment was coatedwith these functional substances according to the following two methods.

The cocktail method: A mixture of two functional substances is added tothe plate. The plate is allowed to stand at 4° C. overnight. The plateis blocked with 2% BSA at 37° C. for 20 minutes and then washed withPBS.

The pre-coating method: A solution of a functional substance having anactivity of binding to a virus is added to the plate. The plate isallowed to stand at 4° C. overnight. The solution is removed. A lamininsolution is added to the plate. The plate is incubated at 37° C. for 2hours, blocked with 2% BSA, and then washed with PBS.

0.5 ml of the solution of the functional substance was used to coat eachwell.

10⁵ L1210 cells and 0.5 ml of Eco-EGFP virus supernatant (10⁵ cfu/ml)were added to the well. The plate was incubated for 24 hours. Afterincubation, the cells were collected by using a cell detachment buffer(Bio Whittaker) and washed. EGFP-expressing cells were analyzed by flowcytometry using FACSVantage™ (Becton Dickinson) at an excitationwavelength of 488 nm and an emission wavelength of 515-545 nm. The genetransfer efficiency (the ratio of EGFP-expressing cells to total cells)was calculated. The experimental results are shown in Tables 2 to 5.TABLE 2 Concentration of laminin added and coating method Functional No5 μg/ml 20 μg/ml substance addition Pre- Pre- 20 μg/ml (80 μg/ml) —coating coating Cocktail BSA (2%) 1.12 5.20 6.55 6.22 H-271 5.41 11.1917.52 9.67 H-296 4.83 5.96 5.51 6.95 CH-271 4.00 6.72 13.73 17.34 CH-2966.48 7.08 6.02 16.77

Gene transfer efficiency in % is indicated. TABLE 3 FunctionalConcentration of laminin added substance No (80 μg/ml) addition 20 μg/ml40 μg/ml 60 μg/ml BSA (2%) 1.36 5.14 4.74 3.82 CH-271 16.89 32.05 24.4523.46 CH-296 17.80 18.79 20.44 19.31

The plate was coated according to the cocktail method. Gene transferefficiency in % is indicated. TABLE 4 Concentration of laminin addedConcentration of No CH-296 added addition 5 μg/ml 10 μg/ml 20 μg/ml Noaddition 0.69 4.09 6.76 6.89 10 μg/ml 4.67 11.81 9.36 7.01 20 μg/ml 5.1611.64 10.57 8.49 40 μg/ml 4.41 10.49 11.52 9.11 80 μg/ml 5.11 10.8711.48 11.10 160 μg/ml  5.19 9.04 11.84 10.88 320 μg/ml  Not done Notdone 10.27 10.54

The plate was coated according to the cocktail method. Gene transferefficiency in % is indicated. TABLE 5 Concentration of laminin addedConcentration of No CH-271 added addition 5 μg/ml 10 μg/ml 20 μg/ml Noaddition 0.69 4.09 6.76 6.89 10 μg/ml 4.61 7.16 6.28 6.34 20 μg/ml 4.7112.98 8.98 5.99 40 μg/ml 3.64 17.32 14.50 8.78 80 μg/ml 3.60 18.30 14.769.15 160 μg/ml  3.52 16.34 17.08 12.67The plate was coated according to the cocktail method. Gene transferefficiency in % is indicated.

As shown in Tables 2 and 3, it was demonstrated that a gene wastransferred into target cells very efficiently, regardless of theimmobilization method, when mouse or human laminin was used incombination with a functional substance having an activity of binding toa virus for gene transfer using a retrovirus. The gene transferefficiency using a plate coated with CH-296 or CH-271 and lamininaccording to the cocktail method was examined. As shown in Tables 4 and5, it revealed that the optimal ratios were 8:1 (e.g., 80 μg/ml: 10μg/ml) for the combination of CH-296/mouse laminin, and 16:1 (e.g., 80μg/ml: 5 μg/ml) for CH-271/mouse laminin, respectively. The genetransfer efficiency increased 2.6-fold and 5.1-fold for CH-296 andCH-271, respectively, as compared with the efficiency of gene transferwithout the addition of laminin.

Example 5 Gene Transfer into Mouse C-Kit-Positive Bone Marrow CellsUsing Laminin

Mouse c-kit-positive bone marrow cells were prepared as follows. Bonemarrow cells collected from a femur of a 6-8 weeks old C3H/He femalemouse (Japan SLC) were subjected to density-gradient centrifugationusing Ficoll-Hypaque (1.0875 g/ml, Pharmacia) to prepare a fractioncontaining low-density mononuclear cells. The cells were washed withPBS, erythrocytes were lysed using Ery-Lysis buffer (155 mM NH₄Cl, 10 mMKHCO₃, 0.1 mM EDTA, pH 7.4), and the cells were washed again with PBS. 1μg/10⁷ cells of an anti-mouse CD117 antibody (Pharmingen) was added tothe resulting bone marrow cells. The mixture was reacted on ice for 30minutes. The cells were washed with PBS containing 5 mM EDTA and 0.5%BSA, and then suspended in the same buffer. 20 μl/10⁷ cells of asecondary antibody conjugated with a microbead (Miltenyi Biotec) wasadded to the cells. The mixture was reacted at 4° C. for 30 minutes. Thecells were washed with and resuspended in the above-mentioned buffer.Cells bound to the microbeads were collected using MACS system (MiltenyiBiotec) to obtain c-kit-positive cells.

Prior to viral infection, the mouse c-kit-positive bone marrow cellswere pre-stimulated in accordance with the method of Luskey et al.[Blood, 80:396-402 (1992)]. Briefly, cells were cultured in α-MEM (BioWhittaker) containing 20% FCS, 20 ng/ml of recombinant mouseinterleukin-3 (Genzyme), 50 ng/ml of recombinant human interleukin-6(Genzyme), 100 ng/ml of recombinant mouse stem cell factor (Genzyme), 50units/ml of penicillin and 50 μg/ml of streptomycin 37° C. for 2 days inthe presence of 5% CO₂.

A 24-well non-treated microplate for cell culture was coated accordingto the cocktail method using a mixture containing mouse laminin at avarying concentration and 80 μg/ml of CH-271. The plate was blocked with2% BSA for 30 minutes, and then washed with PBS. A control plate wasprepared using 2% BSA in place of CH-271. 105 c-kit-positive bone marrowcells and 0.5 ml Eco-EGFP virus supernatant (105 cfu/ml) were added toeach well of the microplate for viral infection. After incubation for 48hours, 0.5 ml of fresh medium was added to the well, and the plate wasincubated for additional 24 hours. After incubation, the cells werecollected by using a cell detachment buffer and washed. The genetransfer efficiency was calculated as described in Example 4. Theresults from two rounds of experiments are shown in Tables 6 and 7.TABLE 6 Concentration of laminin added No addition 10 μg/ml 20 μg/ml BSA0.18 0.25 0.16 CH-271 0.69 3.93 2.64

Gene transfer efficiency in % is indicated. TABLE 7 Concentration oflaminin added No addition 2.5 μg/ml 5 μg/ml 10 μg/ml BSA 1.37 1.80 2.635.38 CH-271 9.95 16.12 15.28 17.00Gene transfer efficiency in % is indicated.

As shown in Tables 6 and 7, it was demonstrated that a very strongeffect of enhancing gene transfer efficiency was also observed whenc-kit-positive bone marrow cells were infected with a retrovirus in aplate coated with mouse laminin and a functional substance having anactivity of binding to a virus, CH-271, according to the cocktailmethod. The efficiency of gene transfer using CH-271 in combination withlaminin increase 5.7-fold at the most as compared with that using CH-271alone.

Furthermore, the same procedure as that described above was carried outusing Eco-EGFP virus supernatant at a titer of 10⁷ cfu/ml. The mean genetransfer efficiency from three rounds of experiments is shown in Table8. It was also demonstrated in this case that the efficiency of genetransfer using a functional substance having an activity of binding to aretrovirus was increased by using laminin in combination. TABLE 8Concentration of laminin added No addition 2 μg/ml 4 μg/ml 6 μg/ml BSA5.88 11.77 19.33 27.09 H-271 25.12 53.39 55.65 56.45 CH-271 43.06 66.8773.67 77.76 CH-296 76.84 81.57 83.30 85.48Gene transfer efficiency in % is indicated.

Example 6 Gene Transfer into CD3-Positive T Cells Derived from MouseSpleen Cells using Laminin

CD3-positive T cells derived from mouse spleen cells were prepared asfollows. Cells were collected from a spleen of a 6-8 weeks old C3H/Hefemale mouse. The cells were passed through a 100-μm mesh (Falcon) toremove residuals. The resulting cells were washed with Hanks' balancedsalt solution (HBSS, Bio Whittaker) containing 10% FCS, erythrocyteswere lysed using Ery-Lysis buffer, and the cells were washed again withHESS. The resulting cells were passed through a 30-μm mesh (MiltenyiBiotec) to remove residuals and then purified using a column forconcentrating CD3-positive T cells (R&D Systems). Mouse CD3-positive Tcells used for viral infection experiments were pre-stimulated asfollows. The cells were cultured for pre-stimulation in a Petri dishonto which an anti-mouse CD3 antibody and an anti-mouse CD28 antibody(both at 1 μg/ml, Pharmingen) had been immobilized. The Petri dishcontained RPMI 1640 medium (Bio Whittaker) supplemented with 10% FCS, 50units/ml of penicillin and 50 μg/ml of streptomycin. The cells werecultured at 37° C. for 2 days in the presence of 5% CO₂.

A 24-well microplate was coated using a mixture containing 20 μg/ml ofmouse laminin and 80 μg/ml of CH-296 as described in Example 5. 10⁵CD3-positive T cells and 0.5 ml Eco-EGFP virus supernatant (10⁵ cfu/ml)were added to each well of the microplate for viral infection for 3hours. RPMI 1640 medium containing 10% FCS, 500 units/ml of recombinantmouse interleukin-1α (Genzyme), 10 ng/ml of recombinant mouseinterleukin-2 (Genzyme), 50 units/ml of penicillin and 50 μg/ml ofstreptomycin was added thereto. The incubation was continued for 48hours. After incubation, the cells were collected by using a celldetachment buffer and washed. The gene transfer efficiency wascalculated as described in Example 4. The results are shown in Table 9.TABLE 9 Functional substance Transfer efficiency (%) BSA (control) 0.83CH-296 8.78 CH-296/mouse laminin 13.20Gene transfer efficiency in % is indicated.

As shown in Table 9, it was demonstrated that the efficiency of genetransfer into mouse CD3-positive T cells was increased by the presenceof laminin.

Example 7 Involvement of Sugar Chain of Laminin Molecule in GeneTransfer

A 96-well microplate was coated using 50 μl/well of a mixture containing5 μg/ml of mouse laminin and 80 μg/ml of CH-271 as described in Example5. The effect of treatment of the plate with various enzymes havingactivities of cleaving sugar chains on gene transfer efficiency wasexamined.

Plates were treated with enzymes as follows: Enzyme solutions containing500 mU/ml O-glycanase (endo-α-N-acetylgalactosaminidase, SeikagakuCorp.), 500 mU/ml endoglycosidase H (endo-β-N-acetylglucosaminidase H,Seikagaku Corp.), 250 mU/ml endo-β-galactosidase (Seikagaku Corp.) and 2mU/ml α-mannosidase (Seikagaku Corp.) in 50 mM citrate-phosphate buffer(pH 5.0) were prepared. An enzyme solution containing 250 mU/mlglycopeptidase F (peptide: N-glycosidase F, Takara Shuzo) in 100 mMtris-hydrochloride buffer (pH 8.6) was prepared. 50 μl each of theenzyme solutions was dispensed in each well for reacting at 37° C. for20 hours. The plate was then washed three times with PBS and then usedfor viral infection experiments.

10⁴ mouse leukemia L1210 cells grown in RPMI 1640 medium supplementedwith 10% FCS, 50 units/ml penicillin and 50 μl/ml streptomycin, and 50μl of PM5neo virus supernatant (10⁴ cfu/ml) were added to each well ofthe microplate. The plate was incubated for 24 hours. The medium waschanged to the same medium containing G418 (Gibco) at a finalconcentration of 0.75 mg/ml. The plate was incubated for additional 48hours. G418-resistance cells were assessed as described in Example 3.The results are shown in Table 10. Table 10 summarizes results fromthree rounds of experiments. TABLE 10 Functional substance Enzymetreatment Absorbance BSA (2%, control) No 0.000 ± 0.030 CH-271 (80μg/ml) No 1.376 ± 0.012 CH-271/laminin No 1.781 ± 0.062 (80 μg/ml:5μg/ml) CH-271/laminin O-Glycanase 1.886 ± 0.071 (80 μg/ml:5 μg/ml)CH-271/laminin Endoglycosidase H 1.214 ± 0.017 (80 μg/ml:5 μg/ml)CH-271/laminin E-β-galactosidase 1.939 ± 0.083 (80 μg/ml:5 μg/ml)CH-271/laminin α-Mannosidase 1.657 ± 0.033 (80 μg/ml:5 μg/ml)CH-271/laminin Glycopeptidase F 1.610 ± 0.036 (80 μg/ml:5 μg/ml)

As shown in Table 10, when CH-271 was used in combination with laminin,the appearance of G418-resistant cells was increased as compared withthe case in which CH-271 was used alone. Treatment of the plate coatedwith laminin with endoglycosidase H completely abolished the genetransfer-promoting effect of laminin. Furthermore, treatment of theplate with α-mannosidase or glycopeptidase F decreased the gene transferefficiency in some degree. According to a report concerning the sugarchains of a laminin molecule [Biochim. Biophysi. Acta, 883:112-126(1986)], most of the sugar chains of the laminin molecule are N-linkedsugar chains which are bound to asparagine. 43 molecules of N-linkedsugar chains are bound to a laminin molecule. Among the sugar chains,high mannose type asparagine-N-linked sugar chains are released bytreatment with endoglycosidase H. The fact that a decrease in genetransfer efficiency was also observed when treated with α-mannosidasesuggests that sugar chains of the laminin molecule play an importantrole. Such sugar chains have a structure containing α1-2- and/orα1-6-bonded mannose, which is cleaved with α-mannosidase, represented by(Mannose)₉-(GlucNAc)₂-Asn and/or (Mannose)₆-(GlucNAc)₂-Asn. As describedabove, it was demonstrated that the gene transfer-promoting effect oflaminin was due to sugar chains of the laminin molecule, in particularhigh mannose type sugar chains.

The involvement of (Mannose)₉-(GlucNAc)₂-Asn in gene transfer wasconfirmed by the following experiments.

1 g of soybean agglutinin prepared from de-fatted soybean flour (Sigma)using Sepharose CL-2B (Pharmacia) to which lactose had been immobilizedwas heat-denatured, and then digested with 20 mg of Actinase E (KakenPharmaceutical) in 20 ml of 50 mM tris-hydrochloride buffer (pH 7.2)containing 10 mM calcium chloride at 37° C. for 2 days. Afterheat-inactivating the enzyme, the mixture was subjected to achromatography using Sephadex G-15 (50 ml) column and Sephadex G-25 (150ml) column to purify (Mannose)₉-(GlucNAc)₂-Asn. FIG. 1 illustrates thestructure of (Mannose)₉-(GlucNAc)₂-Asn from which the asparagine residueis removed.

A microplate to which CH-271 and (Mannose)₉-(GlucNAc)₂-Asn wereimmobilized through covalent bonds was prepared. Briefly, a 96-wellCarboplate (ELISA Carbo-type plate) (Sumitomo Bakelite) was activatedusing 4 mg/ml water-soluble carbodiimide solution at 37° C. for 2 hours,and then washed three times with sterile water. 50 μl each of solutionscontaining 2% BSA or 80 μg/ml of CH-271 as well as(Mannose)₉-(GlucNAc)₂-Asn at a varying concentration was added to eachwell of the activated 96-well Carboplate. The plate was subjected toimmobilization reaction at 37° C. for 2 hours. The plate was blockedusing 0.2% glycine solution at 4° C. for 15 hours and then used for thefollowing gene transfer experiments.

10³ L1210 cells and 0.1 ml Eco-EGFP virus supernatant (10⁶ cfu/ml) wereadded to the well of the microplate. After the plate was incubated for48 hours, 0.1 ml of fresh RPMI 1640 medium containing FCS, penicillinand streptomycin was added to the well. The plate was incubated for anadditional 24 hours. The cells were collected and washed. The genetransfer efficiency was calculated as described in Example 4. The meanresults from two independent experiments are shown in Table 11. TABLE 11Functional Concentration of sugar chain added substance No (80 μg/ml)addition 2.8 μg/ml 5.5 μg/ml 11.1 μg/ml 22.1 μg/ml 44.2 μg/ml 88.5 μg/mlBSA (2%) 1.68 Not Not Not 1.24 1.65 Not done done done done CH-271 26.927.1 29.9 34.7 39.2 52.0 58.7Gene transfer efficiency in % is indicated.

As shown in Table 11, the gene transfer efficiency for the wells onwhich (Mannose)₉-(GlucNAc)₂-Asn and CH-271 had been immobilized wasincreased depending on the concentration of sugar chain used. Thus, itwas confirmed that the sugar chain having the same structure as that ofthe laminin molecule contributed to the increase in gene transferefficiency.

Example 8 Gene Transfer Specific for CD4-Positive Cells Using Anti-CD4Monoclonal Antibody

A 24-well non-treated microplate for cell culture was coated with acombination of 1 μg/ml of an anti-mouse CD4 monoclonal antibody or ananti-mouse CD44 monoclonal antibody (both from Pharmingen) and 80 μg/mlof H-271, CH-271 or CH-296 as described in Example 4. H-271 was coatedaccording to the pre-coating method whereas CH-271 and CH-296 werecoated according to the cocktail method.

0.5 ml of Eco-EGFP virus supernatant (10⁷ cfu/ml) was added to each wellof the microplate. The plate was incubated at 32° C. for 3 hours, andthen washed with RPMI 1640 medium containing 10% FCS, 50 units/ml ofpenicillin and 50 μg/ml of streptomycin. 10⁵ CD3-positive T cellsderived from mouse spleen cells, prepared and pre-stimulated asdescribed in Example 6 were added to the well for viral infection for 3hours. Thereafter, a RPMI 1640 medium containing 10% FCS, 500 units ofrecombinant mouse interleukin-1α, 10 ng/ml of recombinant mouseinterleukin-2, 50 units/ml of penicillin and 50 μg/ml of streptomycinwas added to the well. The plate was incubated for an additional 48hours. After incubation, the cells were collected by using a celldetachment buffer, washed and then stained with an anti-mouse CD4monoclonal antibody (Pharmingen) labeled with phycoerythrin (PE;Pharmingen) and propinium iodide (PI, Sigma). These cells were subjectedto flow cytometry using FACSVantage™ at an excitation wavelength of 488nm and a emission wavelength of 515-545 nm or 562-588 nm totwo-dimensionally analyze CD4 antigen expression and EGFP expression inviable cells. The efficiencies of gene transfer in CD4-positive cellsand CD4-negative cells were calculated. The results are shown in Table12. Table 12 summarizes the results from four rounds of experiments.TABLE 12 Efficiency of Efficiency of transfer into transfer intoFunctional CD4-positive cells CD4-negative cells substance (%) (%) BSA(control)  0.16 ± 0.07  0.11 ± 0.07 Anti-CD4 antibody  0.24 ± 0.19  0.12± 0.04 Anti-CD44 antibody  1.92 ± 0.82  1.95 ± 1.00 H-271 31.02 ± 7.3416.54 ± 4.30 Anti-CD4 antibody/ 58.91 ± 8.11 20.32 ± 4.46 H-271Anti-CD44 antibody/ 56.08 ± 7.53 40.96 ± 7.04 H-271 CH-271 44.63 ± 6.4026.21 ± 5.73 Anti-CD4 antibody/ 64.81 ± 9.74 25.97 ± 1.25 CH-271Anti-CD44 antibody/ 60.29 ± 8.71 44.10 ± 3.56 CH-271 CH-296 48.81 ± 8.7729.45 ± 4.70 Anti-CD4 antibody/ 62.93 ± 6.45 30.84 ± 3.27 CH-296Anti-CD44 antibody/ 56.79 ± 9.87 41.37 ± 1.14 CH-296(Mean ± standard deviation)

As shown in Table 12, when a retroviral infection was carried out in aplate coated with both of a monoclonal antibody and a fibronectinfragment, an effect of enhancing gene transfer efficiency forCD3-positive T cells derived from mouse spleen cells was observed.

Among others, it should be noted that the efficiency of gene transferinto CD4-positive cells was much higher than that into CD4-negativecells when viral infection was carried out using a combination of ananti-CD4 monoclonal antibody and a functional substance having anactivity of binding to a retrovirus. For example, the efficiency of genetransfer into CD4-positive cells using a combination of anti-CD4monoclonal antibody and H-271 was very high (about 60%), while theefficiency of gene transfer into CD4-negative cells was only about 20%.Similar results were observed when CH-271 or CH-296 was used as afibronectin fragment.

On the other hand, CD44 antigen is expressed in 98% of more of bothCD4-positive cells and CD4-negative cells. Therefore, it was expectedthat the gene transfer efficiency would be increased regardless of theexpression of CD4 antigen in the cells when an anti-CD44 monoclonalantibody was used for the retroviral infection as described above. Theresults in Table 12 confirm such expectation.

Example 9 Gene Transfer Specific for CD8-Positive Cells Using Anti-CD8aMonoclonal Antibody

Experiments were carried out as described in Example 8 except that H-271as a functional substance having an activity of binding to a retrovirus,and an anti-mouse CD8a monoclonal antibody (Pharmingen) and ananti-mouse CD44 monoclonal antibody as antibodies were used. Ananti-mouse CD8a monoclonal antibody (Pharmingen) labeled withphycoerythrin (PE; Pharmingen) was used for detecting CD8-positive andCD8-negative cells. The results are shown in Table 13. Table 13summarizes the results from two rounds of experiments. TABLE 13Efficiency of Efficiency of transfer into transfer into FunctionalCD8-positive cells CD8-negative cells substance (%) (%) BSA (control) 0.22 ± 0.08  0.28 ± 0.00 Anti-CD8a antibody  0.36 ± 0.20  0.28 ± 0.02Anti-CD44 antibody  0.98 ± 0.34  0.92 ± 0.20 H-271 20.08 ± 4.71 26.43 ±6.07 Anti-CD8a antibody/ 36.07 ± 1.57 24.42 ± 0.55 H-271 Anti-CD44antibody/ 46.93 ± 0.88 47.16 ± 0.75 H-271(Mean ± standard deviation)

As shown in Table 13, an effect of enhancing the efficiency of genetransfer into CD3-positive T cells derived from mouse spleen cells wasobserved when the combination of an anti-CD8a monoclonal antibody and afibronectin fragment was used.

When the anti-CD8a monoclonal antibody was used, high gene transferefficiency for cells expressing CD8 antigen recognized by the antibodywas observed (Example 8). On the other hand, when a monoclonal antibodyagainst CD44 which is expressed in 98% of more of both CD8-positivecells and CD8-negative cells was used, no difference in gene transferefficiency was recognized between CD8-positive cells and CD8-negativecells.

The experimental results in Examples 8 and 9 are very significant. Theseresults demonstrate that a gene of interest can be transferredspecifically into target cells if a cell population containing targetcells is infected with a retrovirus containing the gene of interest in aculture vessel which has been coated using a mixture (cocktail) of anantibody which specifically binds to the target cells and a functionalsubstance having an activity of binding to the virus.

Example 10 Cell-Specific Gene Transfer Using an Antibody

A 24-well non-treated microplate for cell culture was coated asdescribed in Example 4 according to the cocktail method using 80 μg/mlof CH-271 and 1 μg/ml of one of the monoclonal antibodies againstvarious cell surface antigens (anti-CD4, anti-CD8, anti-CD44,anti-CD49c, anti-CD49d and anti-CD49e antibodies; all from Pharmingen).

K562 (human chronic myelogenous leukemia cell, ATCC CCL-243), HSB-2(human acute lymphoblastic leukemia cell, CCRF-HSB-2, ATCC CCL-120.1),MOLT-3 (human acute lymphoblastic leukemia cell, ATCC CRL-1552) and TF-1(human erythroleukemia cell, ATCC CRL-2003) were used as target cells.FACS analysis was carried out on these cells using labeled monoclonalantibodies to determine the expression of antigens corresponding to theantibodies.

0.5 ml of Ampho-EGFP virus supernatant (1×10⁶ cfu/ml) was added to eachwell of the microplate. The plate was incubated at 32° C. for 3 hours,and then washed with RPMI 1640 medium containing 10% FCS, 50 units/ml ofpenicillin and 50 μg/ml of streptomycin. 1×10⁵ of each of the respectivecells suspended in 1 ml of the medium was added to the well for viralinfection. After incubating for additional 3 days, the cells werecollected by using a cell detachment buffer and washed. The efficiencyof EGFP gene transfer was calculated according to the flow cytometrymethod as described in Example 4.

The results are shown Table 14. The mean results from three independentexperiments are shown. TABLE 14 Cells used HSB-2 MOLT-3 TF-1 K562Transfer CD ag Transfer CD ag Transfer CD ag Transfer CD ag Antibodyeff. exp. eff. exp. eff. exp. eff. exp. used (%) ratio (%) ratio (%)ratio (%) rate None 100 100 100 100 CD4 106.7 − 100.7 +/− 108.8 +/−104.9 − CD8 130.4 ++ 130.4 ++ 107.0 − 116.9 − CD44 173.7 ++ 172.5 ++188.9 +++ 135.1 − CD49c 153.9 +++ 102.7 − 115.6 − 106.3 − CD49d 159.2 ++165.3 +++ 150.3 +++ 97.5 − CD49e 185.5 +++ 127.5 + 128.9 ++ 172.6 +++Gene transfer efficiency (Transfer eff.) is expressed as relative value(%) assuming the efficiency of gene transfer without the addition of anantibody for the respective cells as 100%.CD antigen expression ratios (CD ag exp. ratio) represent the ratios ofpositive cells (%) in FACS measurements as follows:−: 10% or less;+/−: 10-30%;+: 30-60%;++: 60-90%;+++: 90% or more.

As shown in Table 14, the antigen expression ratio correlated with theefficiency of gene transfer using the cocktail method in which CH-271 asa virus-binding substance and the antibody against the antigen on thecell as a cell-binding substance were used.

Furthermore, gene transfer experiments were carried out using 80 μg/mlof polylysine as a functional substance having an activity of binding toa retrovirus in place of CH-271. Monoclonal antibodies and cells used,as well as other experimental conditions, were as described above. Theresults are shown in Table 15. The mean results from three independentexperiments are shown. TABLE 15 Cells used HSB-2 MOLT-3 TF-1 K562Transfer CD ag Transfer CD ag Transfer CD ag Transfer CD ag Antibodyeff. exp. eff. exp. eff. exp. eff. exp. used (%) ratio (%) ratio (%)ratio (%) rate None 100 100 100 100 CD4 103.3 − 104.1 +/− 98.6 +/− 99.4− CD8 116.3 ++ 136.7 ++ 100.8 − 92.4 − CD44 155.5 ++ 144.9 ++ 253.1 +++102.6 − CD49c 160.1 +++ 104.7 − 116.1 − 100.6 − CD49d 138.2 ++ 156.3 +++187.7 +++ 103.1 − CD49e 142.5 +++ 140.0 + 166.1 ++ 129.2 +++Gene transfer efficiency (Transfer eff.) is expressed as relative value(%) assuming the efficiency of gene transfer without the addition of anantibody for the respective cells as 100%.CD antigen expression ratios (CD ag exp. ratio) represent the ratios ofpositive cells (%) in FACS measurements as follows:−: 10% or less;+/−: 10-30%;+: 30-60%;++: 60-90%;+++: 90% or more.

As shown in Table 15, the antigen expression ratio correlated with theefficiency of gene transfer using the cocktail method in whichpolylysine as a virus-binding substance and the antibody against theantigen on the cell as a cell-binding substance were used.

Both series of experimental results as described above demonstrate thata gene can be transferred specifically into target cells of interest bytransferring a gene according to the cocktail method and using anantibody that specifically recognizes an antigen expressed on the targetcell as a cell-binding substance.

Example 11 Gene Transfer into Target Cells Pre-Cultured in MediumContaining Deferoxamine

Human myelocytic leukemia HL-60 cells (ATCC CCL-240) cultured in RPMI1640 medium containing 10% FCS, 50 units/ml of penicillin and 50 μg/mlof streptomycin were transferred into the same medium containingdeferoxamine (Sigma) at a varying concentration on the day before theinfection experiments. The cells were cultured at 37° C. for 20 hours inthe presence of 5% CO₂. The cells were washed with fresh medium withoutdeferoxamine, and then suspended at a concentration of 2×10⁵ cells/mlfor use in the following infection experiments.

0.5 ml of 80 μg/ml CH-271 was added to each well of a 24-wellnon-treated microplate for cell culture. The plate was allowed to standat 4° C. overnight, blocked using 2% BSA for 30 minutes and washed withPBS. 0.5 ml of Ampho-EGFP virus supernatant (10⁶ cfu/ml) was added tothe well of the microplate. The plate was incubated at 32° C. for 3hours and washed with RPMI 1640 medium containing 10% FCS, 50 units/mlof penicillin and 50 μg/ml of streptomycin. 10⁵ of the pre-culturedHL-60 cells were added to the well. The plate was incubated for 48hours. 0.5 ml of RPMI 1640 medium containing 10% FCS, 50 units/ml ofpenicillin and 50 μg/ml of streptomycin was added to the well. The platewas incubated for an additional 24 hours. Thereafter, the gene transferefficiency was determined as described in Example 4. The results areshown in Tables 16 and 17. TABLE 16 Deferoxamine Transfer concentrationFunctional efficiency (μg) substance (%) No addition BSA (control) 0.01No addition CH-271 0.14  6.25 CH-271 0.22 12.5 CH-271 0.27 25 CH-2710.35 50 CH-271 0.71

TABLE 17 Deferoxamine Transfer concentration Functional efficiency (μg)substance (%) No addition BSA (control) 0.02 No addition CH-271 0.25 40CH-271 11.14

As shown in Tables 16 and 17, an increase in gene transfer efficiencywas observed even for HL-60 cells by pre-treating the cells withdeferoxamine for 20 hours. It was known that a gene is transferred intoHL-60 cells with very low efficiency using CH-271 alone.

Example 12 Detection of Presence of Viral Infection-InhibitorySubstances in Culture Supernatant

The TKNeo virus supernatant prepared in Example 2 was diluted with DMEM,a culture supernatant of NIH/3T3 cells (ATCC CRL-1658) or a culturesupernatant of φCRIP cells to a concentration of 312.5 cfu/ml for use inthe following procedures.

0.5 ml of 32 μg/ml CH-296 was added to each well of a 24-wellnon-treated microplate for cell culture. The plate was allowed to standat room temperature for 2 hours, blocked with 2% BSA for 30 minutes andwashed with PBS. 1 ml of the above-mentioned virus supernatant and 2×10⁴NIH/3T3 cells were added to the well of the plate. The plate wasincubated at 37° C. overnight. The cells were then cultured in aselective medium containing 0.75 mg/ml of G418 for 10 days. The numberof colonies formed was counted. The ratio of the number ofG418-resistant colonies to the number of colonies formed in a mediumwithout G418 was defined as gene transfer efficiency. The results areshown in Table 18. TABLE 18 Gene transfer efficiency Diluent (%) DMEM(control) 100 NIH/3T3 cell 20.6 culture supernatant φCRIP cell 15.7culture supernatant

As shown in Table 18, the gene transfer efficiencies were decreased toone fifth or less when the dilution of virus with the culturesupernatant of NIH/3T3 cells or the culture supernatant of φCRIP cellswas used as compared with the efficiency of gene transfer by dilutionwith DMEM. NIH/3T3 cell is the parent strain of many packaging celllines such as φCRIP cell and GP+EmvAm12 cell, which was used to generatethe producer cell for the TKNeo virus vector used in this experiment.The fact that an activity of inhibiting retroviral infection was foundin the culture supernatants of these cells suggests that virussupernatants prepared using similar packaging cells also containinhibitory substances.

Example 13 Removal of Viral Infection-Inhibitory Substance in VirusSupernatant

The following procedures were used to remove the viralinfection-inhibitory substance found in Example 12. The TKNeo virussupernatant prepared in Example 2 was diluted with the culturesupernatant of φCRIP cells to a concentration of 5000 cfu/ml. Thediluted supernatant was further doubly diluted with DMEM for use as asample containing a retrovirus.

1 ml of the virus supernatant was added to each well of a plate coatedwith CH-296 as described in Example 11. The plate was incubated for 1 to5 hours for contacting and binding the virus particles with CH-296. Theplate was then washed three times with PBS. 1 ml of DMEM containing2×10⁴ NIH/3T3 cells was added to the well. As a control, 2×10⁴ NIH/3T3cells were suspended in 1 ml of the above-mentioned virus supernatantand then immediately transferred to the plate coated with CH-296. Theseplates were incubated at 37° C. overnight to allow the virus to infectcells. After infection, the cells were cultured in a selective mediumcontaining 0.75 mg/ml of G418 for 10 days, and the number of coloniesformed were counted. The ratio of the number of G418-resistant coloniesto the number of colonies formed in a medium without G418 was defined asgene transfer efficiency. The results are shown in FIG. 3.

As shown in FIG. 3, higher transfer efficiency was observed at 3 hourswhen virus particles were contacted with and bound to the CH-296 coatedplate as compared with the transfer efficiency for the control group.Thus, it was demonstrated that the activity of inhibiting viralinfection in a virus supernatant could be removed by the proceduresdescribed above.

Example 14 Removal of Sodium Butyrate in Virus Supernatant

Recombinant retrovirus-producer cells obtained by introducing aretrovirus vector plasmid PLEIN into φCRIP cells were cultured in DMEMcontaining 10% CS. When the cells grew to semi-confluence in a 10-cmplate, the medium was changed to 7 ml of RPMI 1640 containing 10% FCS or7 ml of RPMI 1640 containing 5 mM sodium butyrate (Nacalai Tesque) and10% FCS. After the cells were cultured for 24 hours, the supernatantswere filtered through 0.45 μm filters to obtain virus supernatants. Thetiter of the virus supernatant was determined as described in Example 2.The titer of the virus supernatant without sodium butyrate was 3.3×10⁴cfu/ml, whereas the titer of the virus supernatant containing 5 mMsodium butyrate was 2×10⁶ cfu/ml.

Sodium butyrate has the activities of arresting cell cycle to suppresscell growth and of inducing differentiation. Thus, it may have a harmfulinfluence on infected cells. Removal of sodium butyrate contained in avirus supernatant was assessed as follows.

HL-60 cells were used as target cells. 0.5 ml of the above-mentionedvirus supernatant was added to each well of a plate coated with CH-296as described in Example 12. The plate was incubated at 37° C. for 3hours for contacting and binding the virus particles with CH-296. Afterincubation, the plate was washed three times with PBS. 0.5 ml of RPMI1640 medium supplemented with 10% FCS containing 5×10⁴ HL-60 cells wasadded to the well. As a control, 5×10⁴ HL-60 cells were suspended in 0.5ml of the above-mentioned virus supernatant and then immediately addedto the plate coated with CH-296. These plates were incubated at 37° C.overnight to allow the virus to infect cells. After the incubation, 1 mlof RPMI 1640 medium containing 10% FCS was added to the well. The platewas incubated for additional 48 hours. The cell number was then counted.Furthermore, EGFP-expressing cells were detected according to the flowcytometry method as described in Example 4 to analyze the gene transferefficiency. The results are shown in Table 19. TABLE 19 Experimentalgroup/ Cell number Gene transfer virus supernatant (cells/plate)efficiency (%) CH-296 Sodium butyrate: − 2.0 × 10⁵ 2.21 Sodiumbutyrate: + 1.8 × 10⁵ 52.98 Control Sodium butyrate: − 1.7 × 10⁵ 2.74Sodium butyrate: + 4.0 × 10⁵ 35.46

As shown in Table 19, higher gene transfer efficiency was observed forthe control group when a virus supernatant prepared by adding sodiumbutyrate was used, confirming the effectiveness of sodium butyrate inthe virus preparation. However, the viable cell number observed usingthe supernatant containing sodium butyrate was one fourth or less ofthat observed using the supernatant without sodium butyrate, confirmingthat sodium butyrate suppressed cell growth. On the other hand, when thevirus particles were contacted with and bound to the CH-296 coated platebeforehand, the cell growth suppression, although observed for controlgroup using the supernatant containing sodium butyrate, was notobserved. An increase, rather than a decrease, in gene transferefficiency was observed. Thus, it was demonstrated that high genetransfer efficiency could be achieved without the influence of sodiumbutyrate by contacting virus with CH-296 followed by washing.

Next, DEAE-dextran was used to carry out similar experiments.

DEAE-dextran (Sigma) was dissolved at a concentration of 10 mg/ml inPBS. The solution was sterilized by filtering through a 0.22 μm filterand used to coat a plate. 1.1 ml of a mixture obtained by mixing 10volumes of PBS and 1 volume of the DEAE-dextran solution was added toeach well of a 6-well non-treated plate for cell culture (Iwaki Glass).The plate was incubated at 4° C. overnight. The DEAE-dextran solutionwas removed from the plate. 2 ml of 2% BSA solution was added to thewell for treatment for 30 minutes. The plate was washed three times with2 ml/well of PBS. As a control, a plate was prepared by conducting thesame procedures except that PBS was used in place of the DEAE-dextransolution.

A virus supernatant was prepared according to the method in which sodiumbutyrate was added as described above using recombinantretrovirus-producer cells produced by introducing a retrovirus vectorplasmid PLEIN into GP+E86 cells [J. Virol., 62:1120-1124 (1988)]. 1 mlof a virus dilution (1.6×10⁶ cfu/ml) obtained by diluting 1 volume ofthe virus supernatant with 20 volumes of DMEM containing 10% CS wasadded to each well. The plate was incubated at 37° C. for 2 hours, andthen washed three times with 2 ml/well of PBS. 5×10⁴ NIH/3T3 cells wereadded to the well. The plate was incubated at 37° C. for 3 days in thepresence of 5% CO₂. After incubation, the cells were treated withtrypsin and collected. EGFP-expressing cells were analyzed according tothe flow cytometry method as described in Example 4 to determine thegene transfer efficiency. The results are shown in Table 20. TABLE 20Coating Gene transfer efficiency (%) DEAE-dextran 26.7 Control 0.7Gene transfer efficiency represented by the ratio (%) of EGFP-positivecells to total cells is indicated.

As shown in Table 20, it was demonstrated that DEAE-dextran also has anactivity of binding to a retrovirus, and it can be used for the genetransfer method of the present invention.

Example 15 Binding of Functional Substance to Retrovirus UtilizingCentrifugation Method

φCRIP cells into which a retrovirus plasmid, DOL vector, containing aneomycin-resistance gene [Proc. Natl. Acad. Sci. USA, 84:2150-2154(1987)] had been introduced were cultured in DMEM containing 10% CS, 50units/ml of penicillin and 50 μg/ml of streptomycin. A DOL virussupernatant was prepared as follows. Medium in a 10-cm plate in whichthe producer cells had grown to semi-confluence was changed to 5 ml ofDMEM containing 10% CS. After 24 hours, the supernatant was collectedand filtered through a 0.45 μm filter (Millipore). The titer of thevirus supernatant was 8.7×10⁵ cfu/ml.

A centrifugation tube (50 ml polypropylene conical tube, Falcon) usedfor infecting cells with a retrovirus was coated with CH-296 as follows.Briefly, 3 ml of PBS containing 40 μg/ml of CH-296 was slowly placed tothe bottom of the centrifugation tube. The tube was allowed to standupright for incubation at 4° C. for 16 hours. The CH-296 solution wasexchanged for 3.5 ml of PBS containing 2% BSA. The tube was incubatedfor additional 30 minutes at room temperature, and then washed with 5 mlof Hanks' balanced salt solution (HBSS, Gibco).

The DOL retrovirus was bound to the bottom of the centrifugation tubecoated with CH-296 as follows. Briefly, 5 ml of the undiluted DOL virussupernatant, or a 10-fold or 100-fold dilution thereof was placed in thecentrifugation tube. The tube was centrifuged at 2900×g at 25° C. for 3hours to force the retrovirus to bind to CH-296. For comparison, theabove-mentioned virus supernatant was added to a 6-well non-treatedplate for cell culture (Falcon) coated with CH-296 using PBS containing40 μg/ml of CH-296 so as to result in a density of 8 μg/cm². The platewas allowed to stand at 37° C. for 4 hours for binding and used in thefollowing procedures.

Gene transfer into NIH/3T3 cells was carried out using thecentrifugation tube coated with CH-296 to which the retrovirus had beenforced to bind by centrifugation. Briefly, 1×10⁵ NIH/3T3 cells wereplaced in the centrifugation tube coated with CH-296 in which one of theserial dilutions of the virus supernatant had been centrifuged. The tubewas incubated at 37° C. for 3 hours (hereinafter referred to as thecentrifugation method). Alternatively, the above-mentioned microplatewas incubated under the same conditions (hereinafter referred to as thebinding method). As a control, a mixture of the virus supernatant andNIH/3T3 cells was added to the microplate coated with CH-296, and theplate was incubated at 37° C. for 3 hours. The results obtained usingthe last method were defined as those of a conventional infection methodand used for comparison (hereinafter referred to as the supernatantmethod). After incubation, the cells were collected. The efficiency ofgene transfer in the collected cells was determined as described inExample 13. The results are shown in FIG. 4. In FIG. 4, the horizontalaxis represents the dilution rate of the virus supernatant and thevertical axis represents the gene transfer efficiency. The open bars,shaded bars and closed bars represent results obtained using thesupernatant method, the binding method and the centrifugation method,respectively.

As shown in FIG. 4, the efficiency of gene transfer using thecentrifugation method was higher than that using the conventionalsupernatant method or the method in which the virus had beenspontaneously adsorbed to CH-296 prior to the infection (the bindingmethod). Thus, it was demonstrated that more virus particles bound toCH-296 at the bottom of the vessel by forcing the virus to precipitateby centrifugal force. In particular, the effect due to thecentrifugation was remarkable when a diluted virus supernatant was used.

Furthermore, the titers of the virus supernatants collected after thebinding of virus using centrifugation or standing (binding) weremeasured. The results are shown in Table 21. TABLE 21 Virus titerRecovery after Sample (cfu/ml) binding (%) Virus supernatant 8.7 × 10⁵100 (before use) Collected supernatant - 7.8 × 10⁵ 89.4 binding methodCollected supernatant - 7.6 × 10⁴ 8.8 centrifugation method

As shown in Table 21, in the case of standing, the collected supernatanthad about 80 to 90% of the titer of the supernatant before binding. Onthe other hand, the titer of the supernatant after forcing to bind bycentrifugation was one tenth or less of that of the supernatant beforebinding. These results demonstrate that more virus particles were boundto CH-296 by centrifugal force. PBS used to wash the centrifugation tubeafter centrifugation contained about 2% of the original amount of thevirus. In addition, almost the same gene transfer efficiency wasobserved regardless of the presence of the washing step. These resultssuggest that the virus particles were firmly held by CH-296 whencentrifugation was utilized.

Furthermore, the efficiency of gene transfer by the centrifugationmethod was compared with that by a method in which cells are infectedwith a virus during centrifugation.

The efficiency of gene transfer into NIH/3T3 cells by the centrifugationmethod was compared with that by the centrifugation-infection method, inwhich a virus is precipitated by centrifugal force onto cells forinfection (see WO 95/10619). 5 ml of a virus supernatant prepared bydiluting the virus supernatant prepared using GP+E86 cells as describedin Example 14 with a culture supernatant of NIH/3T3 cells to aconcentration of 1×10⁵ cfu/ml was used for the comparison. Briefly, genetransfer was carried out as follows. The above-mentioned virussupernatant was added to a centrifugation tube coated with CH-296. Thetube was centrifuged at 30° C. at 2900×g for 4 hours, and then washedwith PBS. Cells were then added to the tube for infection at 37° C. for4 hours (the centrifugation method). Cell were added to a centrifugationtube coated with CH-296, and cultured for 2 hours. The virus supernatantwas added to the tube. The tube was centrifuged at 30° C. at 2900×g for4 hours for infection (the centrifugation-infection method). In thesemethods, the centrifugation tubes were coated with CH-296 as describedabove, and 1×10⁵ NIH/3T3 cells were used for gene transfer. The cellsafter infection were re-plated into a 60-mm plate, and cultured for 2days. The efficiency of the EGFP gene transfer was then determinedaccording to the flow cytometry method as described in Example 4. Theresults are shown in FIG. 5.

As shown in FIG. 5, it was demonstrated that the efficiency of genetransfer by the centrifugation method was higher than that by thecentrifugation-infection method. This is considered to be becauseinfection-inhibitory substance in the virus supernatant was removed bywashing.

1. A cell culture medium comprising two functional substances: (1) afunctional substance having an activity of binding to a retrovirus; and(2) a functional substance which is an antibody that specifically bindsto a target cell.
 2. The cell culture medium according to claim 1,wherein the functional substance having an activity of binding to theretrovirus is selected from the group consisting of fibronectin,fibroblast growth factor, collagen type V, polylysine and DEAE-dextran,as well as fragments thereof and substances having an equivalentactivity of binding to the retrovirus.
 3. The cell culture mediumaccording to claim 1, wherein the functional substance having anactivity of binding to the retrovirus has an activity of binding to thetarget cell.
 4. The cell culture medium according to claim 1, whereinthe functional substance which is an antibody recognizes CD antigen. 5.The cell culture medium according to claim 1, wherein at least one ofthe two functional substances is immobilized on a substrate.
 6. The cellculture medium according to claim 5, wherein said substrate is a vesselfor cell culture or a particulate substrate.